The present invention relates to a method for detecting microorganisms and, in particular, to a high sensitive method for detecting microorganisms by the flow cytometry.
To determine total viable cell number present in an interested sample is a very important subject in a wide variety of technical fields such as various examinations of food materials possibly contaminated with pathogenic microorganisms and infectious cases or inspection of harmful planktons causing the red tide. On the other hand, when understanding the circulation of substances and energy, it would be very important to recognize the biomass of microorganisms having an important function as decomposers in the ecosystem from the viewpoint of the basic research.
The flow cytometry is a method in which isolated cells are suspended in a medium, then passed through a flow cell at a high speed and subjected to a variety of measurements using optical means. The flow cytometry has become of major interest recently as a quite powerful tool for quickly and correctly counting the number of microorganisms present in a sample, in cooperation with the development of novel fluorescent dyes for staining biological cells. Thus, the flow cytometry has widely and rapidly been used in both the inside and outside of the country for some years ahead as an epoch-making technique that may supersede the colony-counting method and the quantitative ATP-analysis. In this method, it is an absolutely necessary condition that the microorganisms present in the sample are surely stained by the fluorescent dye and the intensity of the fluorescent light rays emitted when the sample passes through a superfine tube is determined by a highly sensitive detector. However, the amount of the fluorescent dye taken by the microorganism widely varies depending on the kind thereof and, in particular, when the amount is considerably lower than the detection limit of the flow cytometry, it has been indicated that it is difficult to correctly determine the viable cell count.
More specifically, it is widely recognized that fluorescence labeling of microorganisms is an effective means to determine the total cell number or how many viable cells exist in a sample. The flow cytometry combined with the fluorescence staining technique has been known as a powerful means to analyze heterogeneous microbial populations (see, for instance, Reference Nos. 7 and 11). Fluorescein diacetate (FDA) and the derivatives thereof are non-fluorescent molecules that defuse into cells and are hydrolyzed by intracellular non-specific esterases to give fluorescent products. The fluorescent products can be accumulated only in those cells that have intact cell membranes, therefore, dead cells with leaky membranes are not stained. Breeuwer et al. (see, for instance, Reference Nos. 5 and 6) reported the precise kinetics of membrane transport and intracellular hydrolysis of FDA and carboxyfluorescein diacetate (CFDA) as determined in studies aimed to optimize fluorescence staining for the detection of yeast cells in food materials by the flow cytometry. However, the fluorescent intensity of labeled cells varies considerably depending on the kinds of strains, probably because of differences in intracellular esterase activities. This may cause inaccuracy in detection of yeast cells in heterogeneous populations by the flow cytometry. Recently, the inventor has found by chance that the accumulation of carboxyfluorescein (CF) or carboxydichlorofluorescein (CDCF) in the cells was facilitated by the application of a hydrostatic pressure of the non-lethal level. Hydrostatic pressure is a thermodynamic variable that acts to decrease the total volume of a system at equilibrium in the case of liquids and solutions. Although the physicochemical basis of the effect of the hydrostatic pressure is well-established (see, for instance, Reference Nos. 2 and 8), the pressure-induced phenomena that occur in living microorganisms have not yet been fully defined.
Accordingly, it is an object of the present invention to provide a highly sensitive method for detecting the number of microorganisms present in a sample, by the flow cytometry.
It is another object of the present invention to provide a method for increasing the amount of fluorescent dye taken into microorganisms.
According to the present invention, there is thus provided a method for detecting microorganisms present in a sample which comprises the steps of
(a) applying a non-lethal hydrostatic pressure to a sample containing microorganisms; and
(b) staining the microorganisms with a fluorescent dye.
According to another aspect of the present invention, there is also provided a method for increasing the uptake of fluorescent dye molecules by microorganisms present in a sample, which comprises the step of applying a non-lethal hydrostatic pressure to the sample containing the microorganism.